The invention relates to polypeptide compounds which possess antagonist properties against bombesin or bombesin-like peptides such as gastrin releasing peptide (GRP), Neuromedin C and the like, hereinafter referred to as bombesin antagonist properties and are of value, for example in the treatment of malignant disease in warm-blooded animals such as man. The invention includes novel polypeptide compounds and processes for their manufacture; novel pharmaceutical compositions containing said polypeptide compounds and processes for the manufacture of medicaments containing them for use in producing a bombesin antagonist effect in warm-blooded animals such as man.
Bombesin is a tetradecapeptide amide which was first isolated from the skin of the frog Bombina - bombina (Anastasi, Erspamer and Bucci, Experientia, 1971, 27, 166). It is known that bombesin is a potent mitogen for mouse Swiss 3T3 fibroblast cells (Rozengurt and Sinnett-Smith, Proc. Natl. Acad. Sci. U.S.A., 1983, 80, 2936) and that it stimulates amylase secretion from guinea pig pancreatic acini (Jensen, Jones, Folkers and Gardner, Nature, 1984, 309, 61). It is also known that bombesin-like peptides are produced and secreted by human small-cell lung cancer (SCLC) cells (Moody, Pert, Gazdar, Carney and Minna, Science, 1981, 214, 1246), that exogenously added bombesin-like peptides can stimulate the growth of human SCLC cells in vitro (Carney, Cuttita, Moody and Minna, Cancer Research, 1987, 47, 821) and that a monoclonal antibody specific for the C-Terminal region of bombesin and GRP can block binding of GRP to its receptors and prevent the growth of human SCLC cells both in vitro and in vivo (Cuttita, Carney, Mulshine, Moody, Fedorko, Fischler and Minna, Nature, 1985, 3167, 823).
GRP which has bombesin-like properties is a widely distributed peptide amide containing 27 amino-acid isolated from the porcine gut (McDonald, Jornvall, Nilsson, Vagne, Ghatei, Bloom and Mutt, Biochem. Biophys. Biophys. Res. Commun., 1979, 90, 227) in which the C-terminal amino acid sequence is almost identical to that of bombesin. Neuromedin C is a decapeptide amide, the structure of which is identical to the last ten amino acids in the C-terminus region of GRP, which has been isolated from the canine small intestine (Reeve, Walsh, Chew, Clark, Hawke and Shively, J. Biol. Chem., 1983, 258, 5582). GRP stimulates a variety of biological responses, including the release of gastrin in the systemic circulation. It also functions as a growth factor in 3T3 mouse fibroblasts and small cell lung cancer (SCLC) cell. So GRP has been proposed to play a direct pathophysiological role in the development of SCLC via an autocrine growth mechanism.
The structures of bombesin, Neuromedin C and Carboxyl-terminal nonapeptide of GRP are shown below:
Bombesin: pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu- Met-NH.sub.2 PA1 Neuromedin C: H-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 PA1 C-terminal nonapeptide of GRP: -Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2 PA1 Q is NH.sub.2 or OQ.sup.1 where Q.sup.1 is hydrogen, C.sub.1-10 alkyl, phenyl or phenyl-C.sub.7-10 -alkyl; PA1 X is hydrogen or a single bond linking to A.sup.2, the acyl residue of an organic acid, or a group of formula R.sup.1 CO-- wherein PA1 X is as above, PA1 Y is --OR.sup.5 or ##STR2## wherein R.sup.5 is hydrogen, C.sub.1-3 alkyl or phenyl; PA1 Dpa is 2,3-diaminopropionic acid PA1 Nal is 3-(2-naphthyl)-alanine PA1 Thi is .beta.-2'-thienylalanine PA1 Tpi is 2,3,4,9 tetrahydro-1 H-pyrido-3,4-b] indole-3-carboxylic acid PA1 Hca is hydrocinnamic acid PA1 Hna is 3-hydroxy-2-naphthoic acid PA1 Hpp is 3-(4-hydroxyphenyl)propionic acid PA1 Mpp is 3-(4-methoxyphenyl)propionic acid PA1 Paa is phenylacetic acid PA1 AC: acyl PA1 Ac: acetyl PA1 AcOH: acetic acid PA1 BOC: tert-butoxycarbonyl PA1 (BOC).sub.2 O: di-tert-butyldicarbonate PA1 BHA: benzhydrylamine PA1 Bzl: benzyl PA1 BSA: bovine serum albumin PA1 DIC: 1,3-diisopropylcarbodiimide PA1 DMEM: Dulbecco's modified Eagle's medium PA1 Et: ethyl PA1 EDTA: ethylene diamine tetraacetic acid PA1 FCBS: fetal calf bovine serium PA1 FMOC: 9-fluorenylmethyloxycarbonyl PA1 For: formyl PA1 HITES: RPMI 16 4D medium plus 10.sup.-8 M hydrocortisone, 5 ul/ml bovine insulin, 10 ug/ml human transferrin, 10.sup.-8 M .beta.-estradiol and 3.times.10.sup.-8 M Na.sub.2 SeO.sub.3 PA1 NOBt: 1-hydroxybenzotriazole PA1 HPLC: high-performance-liquid-chromatography PA1 Me: methyl PA1 MeCN: acetonitrile PA1 MeOH: methyl alcohol PA1 TEA: triethylamine PA1 PBS: phosphate-buffered saline PA1 PGlu: pyroglutamic acid PA1 psi: a pseudo peptide bond of structure CH.sub.2 --NH except where the following residue has a secondary N-terminal in which case the meaning is CH.sub.2 N PA1 TFA: trifluoroacetic acid PA1 Z: benzyloxycarbonyl PA1 (1) wash with CH.sub.2 Cl.sub.2 (3.times.1 min); PA1 (2) deprotection with 50% TFA in CH.sub.2 CH.sub.2 twice for 5 min and 25 min respectively. For peptide resins containing D- or L-Trp or Tpi, deprotection with 50% TFA in CH.sub.2 Cl.sub.2 containing 5% mercaptoethanol and 5% anisol; PA1 (3) wash with CH.sub.2 Cl.sub.2 (6.times.1 min); PA1 (4) neutralization with 10% triethylamine in CH.sub.2 Cl.sub.2 (2.times.3 min) PA1 (5) wash with CH.sub.2 Cl.sub.2 (6.times.1 min); PA1 (6) coupling: PA1 (7) was with CH.sub.2 Cl.sub.2 (2.times.1 min), ethanol (2.times.1 min) and CH.sub.2 Cl.sub.2 (5.times.1 min). PA1 (1) wash with DMF (2.times.1 min) PA1 (2) addition of Boc leucine aldehyde (3equiv.) in DMF containing 1% AcOH; PA1 (3) addition of NaBH.sub.3 CN (3.5 equiv.) in DMF and shaking 60 min; PA1 (4) wash with 50% MeOH (3.times.1 min); 100% MeOH (3.times.1 min); CH.sub.2 Cl.sub.2 (3.times.1 min). PA1 (1) After deprotection and neutralization, wash with CH.sub.2 Cl.sub.2 (3.times.1 min) and DMF (3.times.1 min) PA1 (2) coupling PA1 (3) wash with ethanol (3.times.1 min); DMF (3.times.1 min). PA1 (4) deprotection of Fmoc-group with 50% piperidine in DMF for 30 min. PA1 (5) wash with DMF (6.times.1 min) PA1 (6) another coupling is as described at step (2).
The search for other amphibian bombesin-like peptides led to the isolation of Litorin a nonapeptide (pGlu-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH.sub.2) in the skin of frog from Papua, New Guinea which proves to be the most potent bombesin (Yasukara et al., Chem. Pharm. Bull., 1979, 27, 492). The studies on bombesin analogues showed that a minimum segment of the 9 amino acid residues from 6-14 position of bombesin possessed the full spectrum of bombesin activity.
Several kinds of bombesin antagonists are now known. Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH.sub.2) which has slight amino acid sequence homology with bombesin does not inhibit the binding of bombesin and bombesin-like peptides, but substance P analogues modified by the replacement of several of L-amino acids with D-amino acids such as (D-Arg.sup.1, D-Pro.sup.2, D-Trp.sup.7,9, Leu.sup.11) Substance P and (D-Arg.sup.1, D-Phe.sup.5, D-Trp.sup.7,9, Leu.sup.11) Substance P, (Moody et al., Fed. Proceedings, 1987, 46, 2201) were found to block the secreting of bombesin in pancreatic acinar cells and to antagonize the growth-promoting effects of bombesin in Swiss 3T3 cells. Two types of bombesin antagonists derived from bombesin, for instance, (D-Phe.sup.5, D-Phe.sup.12) bombesin, and [Leu.sup.13 -.sub.psi -Leu.sup.14 ] bombesin (Coy et al., J. Biol. Chem., 1988, 263, 5056 and peptides, 1989, 10, 587) have proved to be potent in vitro and in vivo inhibitors of bombesin response.
Another type of bombesin antagonist revealed by Heimbrook et al., (Bio. Chem., 1989, 264, 11258) is N-acetyl-GRP(20-26) and its analogous wherein the C-terminal methionine residue is deleted from GRP(20-27) analogues. Recently, Coy [J. Biol. Chem. 264, 1989, 25, 14691] reported that some short chain bombesin antagonists based on Litorin sequence such as [D-Phe.sup.6, Leu.sup.13 -.sub.psl -Phe.sup.14 ] bombesin-(6-14) and [D-Phe.sup.6, Leu.sup.13 -.sub.psi -Leu.sup.14 ] bombesin-(6-14) exhibited much more potency than their corresponding parent peptide [Leu.sup.13 -.sub.psi -Leu.sup.14 ] bombesin.